U.S. patent number 5,268,384 [Application Number 07/817,039] was granted by the patent office on 1993-12-07 for inhibition of angiogenesis by synthetic matrix metalloprotease inhibitors.
Invention is credited to Richard E. Galardy.
United States Patent |
5,268,384 |
Galardy |
December 7, 1993 |
Inhibition of angiogenesis by synthetic matrix metalloprotease
inhibitors
Abstract
Synthetic mammalian matrix metalloprotease inhibitors are useful
in controlling angiogenesis. These compounds are thus useful in
controlling the growth of tumors and in controlling neovascular
glaucomas.
Inventors: |
Galardy; Richard E. (Suilford,
CT) |
Appl.
No.: |
07/817,039 |
Filed: |
January 7, 1992 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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747751 |
Aug 20, 1991 |
5239078 |
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747752 |
Aug 20, 1991 |
5189178 |
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615798 |
Nov 21, 1990 |
5183900 |
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Current International
Class: |
C07D 209/20 ();
A61K 031/405 () |
Field of
Search: |
;548/495 ;514/419 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0126974 |
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Dec 1984 |
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EP |
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0159396 |
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Oct 1985 |
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EP |
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0276436 |
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Aug 1988 |
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EP |
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0424193 |
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Apr 1991 |
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EP |
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57-058626 |
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Apr 1982 |
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JP |
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WO88/06890 |
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Sep 1988 |
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WO |
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WO91/11193 |
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Aug 1991 |
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WO |
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Other References
Mullins et al., Biochim. Biophys. Acta (1983) 695:177-214. .
Reich et al., Cancer Res. (1988) 48:3307-3312. .
Nishino et al., Biochemistry (1979) 18:4340-4347. .
Nishino et al., Biochemistry (1978) 17:2846-2850..
|
Primary Examiner: Springer; David B.
Attorney, Agent or Firm: Cagan; Felissa H. Giotta; Gregory
J.
Government Interests
This invention was made with government support under grant HL27368
awarded by the National Institutes of Health. The government has
certain rights in the invention.
Parent Case Text
This application is a continuation-in-part of U.S. Ser. No.
07/747,751, filed Aug. 20, 1991; now U.S. Pat. No. 5,239,078, U.S.
Ser. No. 07/747,752, filed Aug. 20, 199 now U.S. Pat. No.
5,184,178; and 07/615,798, filed Nov. 21, 1990, now U.S. Pat. No.
5,183,900.
Claims
I claim:
1. A method to inhibit angiogenesis which method comprises
contacting a tissue in which unwanted angiogenesis is occurring
with an effective amount of a synthetic mammalian matrix
metalloprotease inhibitor, wherein said inhibitor is selected from
the group consisting of inhibitors of the formulae: ##STR14##
wherein R is H or lower alkyl (1-6C); and wherein R.sup.4 is
(3-indolyl)methylene, and the pharmaceutically acceptable amides
thereof.
2. The method of claim 1 wherein the inhibitor is NHOHCOCH.sub.2
CH(i-Bu)CO-L-Trp-NHMe.
3. A composition for inhibition of angiogenesis which composition
comprises at least one synthetic mammalian matrix metalloprotease
inhibitor in admixture with at least one pharmaceutically
acceptable excipient, wherein said inhibitor is selected from the
group consisting of inhibitors of the formulae: ##STR15## wherein R
is H or lower alkyl (1-6C); and wherein R.sup.4 is
(3-indolyl)methylene, and the pharmaceutically acceptable amides
thereof.
4. The composition of claim 3 wherein the inhibitor is
NHOHCOCH.sub.2 CH(i-Bu)CO-L-Trp-NHMe.
5. The method of claim 1 wherein said inhibitor is of the formula:
##STR16## wherein R is H or lower alkyl (1-6C); and wherein R.sup.4
is (3-indolyl)methylene, and the pharmaceutically acceptable amides
thereof.
6. The method of claim 1 wherein said inhibitor is of the formula:
##STR17## and wherein R.sup.4 is (3-indolyl)methylene, and the
pharmaceutically acceptable amides thereof.
7. The composition of claim 3 wherein said inhibitor is of the
formula: ##STR18## wherein R is H or lower alkyl (1-6C); and
wherein R.sup.4 is (3-indolyl)methylene, and the pharmaceutically
acceptable amides thereof.
8. The composition of claim 3 wherein said inhibitor is of the
formula: ##STR19## and wherein R.sup.4 is (3-indolyl)methylene, and
the pharmaceutically acceptable amides thereof.
9. A method to inhibit angiogenesis which method comprises
contacting a tissue in which unwanted angiogenesis is occurring
with an effective amount of a synthetic mammalian matrix
metalloprotease inhibitor, wherein the inhibitor is of the
formula:
and wherein R.sup.4 is (3-indolyl)methylene, and the
pharmaceutically acceptable amides thereof.
10. The method of claim 9 wherein the inhibitor is NHOHCOCH.sub.2
CH(i-Bu)CO-L-Trp-NHCHMePh.
11. The method of claim 9 wherein the inhibitor is HOOCCH.sub.2
CH(i-Bu)CO-L-Trp-NHCHMePh.
12. A composition for inhibition of angiogenesis which composition
comprises at least one synthetic mammalian matrix metalloprotease
inhibitor in admixture with at least one pharmaceutically
acceptable excipient, wherein said inhibitor is of the formula:
and wherein R.sup.4 is (3-indolyl)methylene, and the
pharmaceutically acceptable amides thereof.
13. The composition of claim 12 wherein the inhibitor is
NHOHCOCH.sub.2 CH(i-Bu)CO-L-Trp-NHCHMePh.
14. The method of claim 12 wherein the inhibitor is HOOCCH.sub.2
CH(i-Bu)CO-L-Trp-NHCHMePh.
Description
TECHNICAL FIELD
The invention relates to synthetic compounds that are known to
inhibit matrix metalloproteases and to their ability to inhibit
angiogenesis. More specifically, the invention concerns treating
conditions associated with unwanted angiogenesis using these matrix
metalloprotease inhibitors.
RELEVANT ART
Angiogenesis is defined as the growth of new blood vessels, in
particular, capillaries. The ingrowth of such capillaries and
ancillary blood vessels is essential for tumor growth and is thus
an unwanted physiological response which encourages the spread of
malignant tissue and metastases. Inhibition of angiogenesis is
therefore envisioned as a component of effective treatment of
malignancy Neovascularization of the eye is a major cause of
blindness. One form of this condition, proliferative diabetis
retinopathy, results from diabetes; blindness can also be caused by
neovascular glaucoma. Inhibition of angiogenesis is useful in
treating these conditions also.
PCT application WO 91/11193, published Jan. 25, 1991 describes the
isolation of a collagenase inhibitor from cartilage which inhibits
the formation of blood vessels. This composition, designated
cartilage-derived inhibitor (CDI), is reported to inhibit
tumor-induced angiogenesis in the rabbit corneal pocket assay and
to inhibit capillary tube formation. It is further speculated that
other collagenase inhibitors such as peptides or antibodies
immunoreactive with collagenase will also have the ability to
inhibit blood vessel formation.
In addition, EP application 424,193 published Apr. 24, 1991,
describes the activity of actinonin as an angiogenesis inhibitor.
Actinonin is an antibiotic produced by a particular strain of
Streptomyces and is a modified peptide structure.
As disclosed in the two foregoing applications, unwanted levels of
angiogenesis are present not only in association with tumor growth,
but also are the cause of blindness resulting from diabetic
retinopathy and other ocular pathologies.
The present invention supplies alternative compounds that are
useful in inhibition of angiogenesis, and which can be supplied as
synthetic compounds in isolated and purified form.
DISCLOSURE OF THE INVENTION
The methods and compositions of the invention for control of
angiogenesis comprise, as active ingredient, at least one synthetic
metalloprotease inhibitor. Some members of this class of compounds
are known in the art; others are described and claimed in U.S. Ser.
No. 07/747,751, filed Aug. 20, 1991; U.S. Ser. No. 747,752, filed
Aug. 20, 1991; and U.S. Ser. No. 07/615,798, filed Nov. 21, 1990,
the disclosures of which are incorporated herein by reference.
A summary of the art-known synthetic matrix metalloprotease
inhibitors is found in EP application 423,943 published Apr. 24,
1991. This application assembles the structures of the synthetic
matrix metalloproteases known in the art and claims their use in
the treatment of demyelinating diseases of the nervous system. The
present invention is directed to the use of these compounds, as
well as those disclosed in the above-referenced U.S. applications,
in inhibiting angiogenesis.
Thus, in one aspect, the invention is directed to a method to
inhibit angiogenesis which method comprises administering, to the
site at which unwanted angiogenesis occurs, an effective amount of
at least one synthetic mammalian matrix metalloprotease inhibitor.
In other aspects, the invention is directed to compositions useful
in inhibiting angiogenesis containing, as active ingredient, at
least one synthetic mammalian matrix metalloprotease inhibitor.
MODES OF CARRYING OUT THE INVENTION
The angiogenesis inhibitory compounds of the invention are
synthetic inhibitors of mammalian matrix metalloproteases. Matrix
metalloproteases include without limitation human skin fibroblast
collagenase, human skin fibroblast gelatinase, purulent human
sputum collagenase and gelatinase, and human stromelysin. These are
zinc-containing metalloprotease enzymes, as are the
angiotensin-converting enzymes and the enkephalinases. As used
herein, "mammalian matrix metalloprotease" means any
zinc-containing enzyme found in mammalian sources that is capable
of catalyzing the breakdown of collagen, gelatin or proteoglycan
under suitable assay conditions.
Appropriate assay conditions can be found, for example, in U.S.
Pat. No. 4,743,587, which references the procedure of Cawston, et
al., Anal Biochem (1979) 99:340-345, use of a synthetic substrate
is described by Weingarten, H., et al., Biochem Biophys Res Comm
(1984) 139:1184-1187. Any standard method for analyzing the
breakdown of these structural proteins can, of course, be used. The
matrix metalloprotease enzymes referred to in the herein invention
are all zinc-containing proteases which are similar in structure
to, for example, human stromelysin or skin fibroblast
collagenase.
The ability of candidate compounds to inhibit matrix
metalloprotease activity can, of course, be tested in the assays
described above. Isolated matrix metalloprotease enzymes can be
used to confirm the inhibiting activity of the invention compounds,
or crude extracts which contain the range of enzymes capable of
tissue breakdown can be used.
Specifically, assay of inhibition activity can be conducted as
follows. Inhibitors may be assayed against crude or purified human
skin fibroblast collagenase using the synthetic thiol ester
substrate at pH 6.5 exactly as described by Kortylewicz &
Galardy, J Med Chem (1990) 33:263-273, at a collagenase
concentration of 1-2 nM. The candidate inhibitors are tested for
their ability to inhibit crude collagenase and gelatinase from
human skin fibroblasts, crude collagenase and gelatinase from
purulent human sputum in this assay. The results may be set forth
in terms of Ki, i.e., the calculated dissociation constant for the
inhibitor complex with enzyme. Ki values for effective inhibitors
are .ltoreq.500 nM for purified enzyme in this assay. For purified
human skin collagenase, excellent inhibitors show Ki values of
.ltoreq.10 nM. Assays for inhibition of human stromelysin are
conducted as described by Teahan, J., et al., Biochemistry (1989)
20:8497-8501.
The synthetic compounds that are successful in these assays for
mammalian matrix metalloprotease inhibition are generally small
molecules containing at least one amide bond and have a variety of
sidechain substituents. Examples of such compounds known in the art
are given, as set forth above, in EP application publication no.
423,943, incorporated herein by reference.
Other suitable inhibitors are of the formula: ##STR1## wherein
each R.sup.1 is independently H or alkyl (1-8C) and R.sup.2 is
alkyl (1-8C) or wherein the proximal R.sup.1 and R.sup.2 taken
together are --(CH.sub.2).sub.p -- wherein p=3-5;
R.sup.3 is H or alkyl (1-4C);
R.sup.4 is fused or conjugated unsubstituted or substituted
bicycloaryl methylene;
n is 0, 1 or 2;
m is 0 or 1; and
X is OR.sup.5 or NHR.sup.5, wherein R.sup.5 is H or substituted or
unsubstituted alkyl (1-12C), aryl (6-12C), aryl alkyl (6-16C);
or
X is an amino acid residue or amide thereof; or
X is the residue of a cyclic amine or heterocyclic amine; and
R.sup.6 is H or lower alkyl (1-4C) and R.sup.7 is H, lower alkyl
(1-4C) or an acyl group, and
wherein the --CONR.sup.3 -- amide bond shown is optionally replaced
by a modified isosteric bond, such as --CH.sub.2 NR.sup.3 --,
--CH.sub.2 CHR.sup.3 --, --CH.dbd.CR.sup.3 --, --COCHR.sup.3 --,
--CHOHCHR.sup.3 --, --NR.sup.3 CO--, --CF.dbd.CR.sup.3 --, and the
like.
Other compounds useful in the method of the invention include
compounds of the formulas ##STR2## wherein
each R.sup.1 is independently H or alkyl (1-8C) and R.sup.2 is
alkyl (1-8C) or wherein the proximal R.sup.1 and R.sup.2 taken
together are --(CH.sub.2).sub.p -- wherein p=3-5;
R.sup.3 is H or alkyl (1-4C);
R.sup.4 is fused or conjugated unsubstituted or substituted
bicycloaryl methylene;
n is 0, 1 or 2;
m is 0 or 1; and
X is OR.sup.5 or NHR.sup.5, wherein R.sup.5 is H or substituted or
unsubstituted alkyl (1-12C), aryl (6-12C), aryl alkyl (6-16C);
or
X is an amino acid residue or amide thereof; or
X is the residue of a cyclic amine or heterocyclic amine;
Y is selected from the group consisting of R.sup.7 ONR.sup.6
CONR.sup.6 --, R.sup.6.sub.2 NCONOR.sup.7 --, and R.sup.6
CONOR.sup.7 --, wherein each R.sup.6 is independently H or lower
alkyl (1-4C); R.sup.7 is H, lower alkyl (1-4C) or an acyl group,
and
wherein the --CONR.sup.3 -- amide bond shown is optionally replaced
by a modified isosteric bond, such as --CH NR.sup.3 --, --CH.sub.2
CHR.sup.3 --, --CH.dbd.CR.sup.3 --, --COCHR.sup.3 --,
--CHOHCHR.sup.3 --, --NR.sup.3 CO--, --CF.dbd.CR.sup.3 --, and the
like.
"Alkyl" has its conventional meaning as a straight chain, branched
chain or cyclic saturated hydrocarbyl residue such as methyl,
ethyl, isobutyl, cyclohexyl, t-butyl or the like. The alkyl
substituents of the invention are of the number of carbons noted
which may be substituted with 1 or 2 substituents. Substituents are
generally those which do not interfere with the activity of the
compound, including hydroxyl, "CBZ," amino, and the like. Aryl
refers to aromatic ring systems such as phenyl, naphthyl, pyridyl,
quinolyl, indolyl, and the like; aryl alkyl refers to aryl residues
linked to the position indicated through an alkyl residue. In all
cases the aryl portion may be substituted or unsubstituted. "Acyl"
refers to a substituent of the formula RCO-- wherein R is alkyl or
arylalkyl as above-defined. The number of carbons in the acyl group
is generally 1-15; however as the acyl substitute is readily
hydroxylized in vivo the nature of the group is relatively
unimportant. "Cyclic amines" refer to those amines where the
nitrogen is part of a heterocyclic ring, such as piperidine,
"heterocyclic amines" refer to such heterocycles which contain an
additional heteroatom, such as morpholine.
In the compounds of formulas 1 and 3, preferred embodiments for
R.sup.1 and R.sup.2 include those Wherein each R.sup.1 is H or Me
and R.sup.2 is alkyl of 3-8C, especially isobutyl, 2-methyl butyl,
or isopropyl. Especially preferred is isobutyl. Preferred also are
those compounds of all of formulas 1-4, wherein n=1 or m=1.
In all of formulas 1-4, preferred embodiments of R.sup.3 are H and
methyl, especially H.
R.sup.4 is a fused or conjugated bicyclo aromatic system linked
through a methylene group to the molecule. By "fused or conjugated
bicyclo aromatic system" is meant a two-ringed system with aromatic
character which may, further, contain one or more heteroatoms such
as S, N, or O. When a heteroatom such as N is included, the system
as it forms a part of formulas 1-4, may contain an acyl protecting
group (1-5C) attached to the nitrogen. Representative bicyclo fused
aromatic systems include naphthyl, indolyl, quinolinyl, and
isoquinolinyl. Representative conjugated systems include biphenyl,
4-phenylpyrimidyl, 3-phenylpyridyl and the like. In all cases, any
available position of the fused or conjugated bicyclic system can
be used for attachment through the methylene. The fused or
conjugated aromatic system may further be substituted by 1-2 alkyl
(1-4C) residues and/or hydroxy or any ring nitrogens may be
acylated. Preferred acylation is acetylation.
Preferred embodiments of R.sup.4 include 1-(2-methyl
naphthyl)methylene; 1-quinolyl methylene; 1-naphthyl methylene;
2-naphthyl methylene; 1-isoquinolyl methylene; 3-isoquinolyl
methylene; 3-thionaphthenyl methylene; 3-cumaronyl methylene;
3-(5-methylindolyl)methylene; 3-(5-hydroxyindolyl)methylene;
3-(2-hydroxyindolyl)methylene; biphenyl methylene; and
4-phenylpyrimidyl methylene; and the substituted forms thereof.
Many of these substituents as part of an amino acid residue are
described in Greenstein and Winitz, "Chemistry Of the Amino Acids"
(1961) 3:2731-2741 (John Wiley & Sons, NY).
A particularly preferred embodiment of R.sup.4 is
3-indolylmethylene or its N-acylated derivative--i.e., that
embodiment wherein the "C-terminal" amino acid is a tryptophan
residue or a protected form thereof. A preferred configuration at
the carbon to which R.sup.4 is bound is that corresponding to
L-tryptophan.
Preferred embodiments of X are those of the formula NHR.sup.5
wherein R.sup.5 is H, substituted or unsubstituted alkyl (1-12C) or
aryl alkyl (6-12C). Particularly preferred substitutions on R.sup.5
are a hydroxyl group, or a phenylmethoxycarbamyl (CBZ) residue. In
addition, the compound may be extended by embodiments wherein X is
an additional amino acid residue, particularly a glycyl residue,
which may also be amidated as described.
In general, the compounds that are hydroxamates are obtained by
converting a carboxylic acid or ester precursor of the formulas
##STR3## wherein R is H or alkyl (1-6C) to the corresponding
hydroxamates by treating these compounds or their activated forms
with hydroxylamine under conditions which effect the
conversion.
With respect to starting materials, the components forming the
--NR.sup.3 --CHR.sup.4 COX moiety are readily available in the case
of tryptophan and its analogs as esters or amides. As set forth
above, many analogous fused bicyclo aromatic amino acids are
described by Greenstein and Winitz (supra). Amino acids
corresponding to those wherein R.sup.4 is 1-(2-methyl
naphthyl)methylene; 1 -quinolyl-methylene; 1-naphthyl methylene;
1-isoquinolyl methylene; and 3-isoquinolyl methylene can be
prepared from the bicyclo aromatic methylene halides using the
acetamido malonic ester synthesis of amino acids, as is well
understood in the art. The methylene halides themselves can be
prepared from their corresponding carboxylic acids by reduction
with lithium aluminum hydride and bromination of the resulting
alcohol with thionyl bromide.
In general, the hydroxylamine reagent is formed in situ by mixing
the hydroxylamine hydrochloride salt with an excess of KOH in
methanol and removing the precipitated potassium chloride by
filtration. The filtrate is then stirred with the precursor
activated carboxylic acid or ester of formula 5 or 6 for several
hours at room temperature, and the mixture is then evaporated to
dryness under reduced pressure. The residue is acidified, then
extracted with a suitable organic solvent such as ethyl acetate,
the extract washed with aqueous potassium bisulfate and salt, and
then dried with a solid drying agent such as anhydrous magnesium
sulfate. The extract is then again evaporated to dryness and
crystallized.
The substituted forms of the hydroxamate which include --NHOR.sup.7
are synthesized in an analogous manner but substituting H.sub.2
NOR.sup.7, wherein R.sup.7 is lower alkyl or acyl (1-4C) for
hydroxylamine per se. The resulting O-alkyl or acyl hydroxamate can
then be further alkylated, if desired, to obtain the R.sup.7
ONR.sup.6 -- derivative of the carboxylic acid. Similarly,
HNR.sup.6 OH may be reacted with the carboxylic acid to obtain the
HONR.sup.6 -- derivative. HNCH.sub.3 OH and H.sub.2 NOCH.sub.3 are
commercially available.
To prepare the starting materials of formulas 5 and 6, the
monoesterified carboxylic acid of the formula ##STR4## or of the
formula ##STR5## is reacted with the acid of the formula
wherein X is other than OH under conditions wherein the
condensation to form the amide bond occurs. Such conditions
typically comprise mixture of the two components in a nonaqueous
anhydrous polar aprotic solvent in the presence of base and a
condensing agent such as a carbodiimide. Thus, the formation of the
amide linkage can be catalyzed in the presence of standard
dehydration agents such as the carbodiimides, for example
dicyclohexyl carbodiimide, or N, N-carbonyl diimidazole. The
product is then recovered as a mixture of diastereomers of formula
5 or 6. This mixture is preferably used for the conversion to the
hydroxamate and one of the resulting diastereomers is crystallized
directly from the product mixture. Alternatively, the diastereomers
are separated by flash chromatography before conversion to the
hydroxamate and recovered separately. This process is less
preferred as compared to the process wherein separation of the
diastereomers is reserved until the final product is obtained.
In the notation used in the examples, the "A" isomer is defined as
that which migrates faster on TLC; the "B" isomer as that which
migrates more slowly. When the "L" form of tryptophan or other
amino acid containing a fused bicycloaromatic ring system is used
as the residue, and R.sup.1 is H, in general, the "A" form is that
which contains the corresponding configuration at the carbon
containing the R.sup.2 substituent (providing that is the only
other center of asymmetry) in the final hydroxamate product.
However, in Example 2, below, where D-tryptophan is included in the
composition, the "B" isomer contains what would correspond to an
"L" configuration at the carbon containing R.sup.2 in the compounds
of formula 1.
When R.sup.6 and/or R.sup.7 =alkyl, the corresponding O- or N-alkyl
hydroxylamine is reacted with the methyl ester 4A as performed for
unsubstituted hydroxylamine in Example 1. Alternatively, the methyl
ester 4A can be saponified to its corresponding carboxylic acid and
activated with oxalyl chloride or other condensing agent. The alkyl
hydroxylamine can then be reacted with the activated carboxylic
acid to give the O- or N-substituted hydroxamic acid. O- and
N-methylhydroxylamine can be purchased from the Aldrich Chemical
Company.
Other N-alkyl hydroxylamines can be synthesized by conversion of
aliphatic aldehydes to their oximes, followed by reduction to the
N-alkyl hydroxylamine with borane-pyridine complex in the presence
of 6N HCl (Kawase, M. and Kikugawa, Y.J., Chem Soc, Perkin Trans
(1979) 1:643. Other O-alkyl hydroxylamines can be synthesized by
the general methods given by Roberts, J.S., "Derivatives of
Hydroxylamine," Chapter 6.4 in Barton, D., et al., eds.,
Comprehensive Organic Chemistry (1979) 2:187-188 (Pergamon Press,
Oxford). The two general methods employed are displacement by
R.sup.7 O-- of a leaving group from hydroxylamine sulfonic acid or
chloramine, and O-alkylation of a hydroxamic acid with R.sup.7 --X
followed by hydrolysis: ##STR6##
For R.sup.7 =acyl, a hydroxamic acid of this invention can be
acylated with an acid chloride, anhydride, or other acylating agent
to give the compounds of this class.
In some cases the derivatized maleic and succinic acid residues
required for synthesis of the invention compounds are commercially
available. If not, these can readily be prepared, in embodiments
wherein R.sup.1 is H or alkyl (1-8C) by reaction of a
2-oxocarboxylic ester of the formula R.sup.2 COCOOR, in a Wittig
reaction with an alkyl triphenylphosphoranylidene acetate or
.alpha.-triphenylphosphoranylidene alkanoate. The methyl acetate or
alkanoate is preferred, but any suitable ester can be employed.
This reaction is conducted in a nonaqueous, nonpolar solvent
usually at room temperature. The resultant compound is of the
formula ROOCCR.sup.1 .dbd.CR.sup.2 COOR', wherein R and R' are
residues of esterifying alkyl or arylalkyl alcohols.
If the compounds of formula 6 are desired, this product is
condensed with the appropriate tryptophan or analogous derivative;
if the compounds of formula 5 are desired, the intermediate is
reduced using hydrogen with a suitable catalyst. The sequence of
reactions to obtain those embodiments wherein R.sup.1 is H or
alkyl, n is 1 and m is 0, and R.sup.2 is alkyl are shown in
Reaction Scheme 1. ##STR7##
For those embodiments wherein R.sup.1 and R.sup.2 taken together
are (CH.sub.2).sub.p, the compounds of the invention are prepared
analogously to the manner set forth in Reaction Scheme 1, except
that the intermediate of the formula ROOCCHR.sup.1 CHR.sup.2 COOH
is prepared from the corresponding 1,2-cycloalkane dicarboxylic
acid--i.e., 1,2-cyclopentane dicarboxylic acid anhydride;
1,2-cyclohexane dicarboxylic anhydride or 1,2-cycloheptane
dicarboxylic anhydride.
For compounds wherein --CONR.sup.3 -- is in modified isosteric
form, these forms can be prepared by methods known in the art. The
following references describe preparation of peptide analogs which
include these alternative-linking moieties: Spatola, A.F., Vega
Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone
Modifications" (general review); Spatola, A.F., in "Chemistry and
Biochemistry of Amino Acids Peptides and Proteins," B. Weinstein,
eds., Marcel Dekker, New York, p. 267 (1983) (general review);
Morley, J.S., Trends Pharm Sci (1980) pp. 463-468 (general review);
Hudson, D., et al., Int J Pept Prot Res (1979) 14:177-185
(--CH.sub.2 NR.sup.3 --, --CH.sub.2 CHR.sup.3 --); Spatola, A.F.,
et al., Life Sci (1986) 38:1243-1249 (--CH.sub.2 --S); Hann, M.M.,
J Chem Soc Perkin Trans I (1982) 307-314 (--CH--CR.sup.3 --, cis
and trans); Almquist, R.G., et al., J Med Chem (1980) 23:1392-1398
(--COCHR.sup.3 --); Jennings-White, C., et al., Tetrahedron Lett
(1982) 23:2533 (--COCHR.sup.3 --); Szelke, M., et al., European
Application Ep 45665 (1982) CA:97:39405 (1982) (--CH(OH)CHR.sup.3
--); Holladay, M.W., et al., Tetrahedron Lett (1983) 24:4401-4404
(--C(OH)CH.sub.2 --); and Hruby, V.J., Life Sci (1982) 31:189-199
(--CH.sub.2 --S--).
Preferred compounds of formula (1) or (2) include:
HONHCOCH.sub.2 CH(n-hexyl)-CO-L-Trp-NHMe;
HONHCOCH.sub.2 CH(n-pentyl)-CO-L-Trp-NHMe;
HONHCOCH.sub.2 CH(i-pentyl)-CO-L-Trp-NHMe;
HONHCOCH.sub.2 CH(ethyl)-CO-L-Trp-NHMe;
HONHCOCH.sub.2 CH(ethyl)-CO-L-Trp-NHCH.sub.2 CH.sub.3 ;
HONHCOCH.sub.2 CH(ethyl)-CO-L-Trp-NHCH.sub.2 CH.sub.2 OH;
HONHCOCH.sub.2 CH(ethyl)-CO-L-Trp-NHcyclohexyl;
MeONHCOCH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
EtONMeCOCH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
MeONHCOCH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
EtONMeCOCH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
HONHCOCH.sub.2 CH(i-Bu)CO-L-Trp-NHMe;
HONHCOCH.sub.2 CH(i-Bu)CO-L-N-MeTrp-NHMe;
HONHCOCH.sub.2 CH(i-Bu)CO-L-Trp-NH(CH.sub.2).sub.2 OH;
HONHCOCH.sub.2 CH(i-Bu)CO-L-Trp-NH(S)CHMePh;
HONHCOCH.sub.2 CH(i-Bu)CO-L-Trp-NH(CH.sub.2).sub.6 NH-CBZ;
HONHCOCH.sub.2 CH(i-Bu)CO-L-Ala(2-naphthyl)NHMe;
HONHCOCH.sub.2 CH(i-Bu)CO-L-Trp-NH(CH.sub.2).sub.4 CH.sub.3 ;
HONHCOCH.sub.2 CH(i-Bu)CO-L-Trp-piperidine;
HONHCOCH.sub.2 CH(i-Bu)CO-L-Trp-NH(CH.sub.2).sub.11 CH.sub.3 ;
HONHCOCH.sub.2 CH(i-Bu)CO-L-Trp-NHcyclohexyl;
HONHCOCH.sub.2 CH(i-Bu)-L-Trp-OH;
HONMeCOCH.sub.2 CH(i-Bu)CO-L-Trp-NHMe;
HONEtCOCH.sub.2 CH(i-Bu)CO-L-Trp-NHMe;
CH.sub.3 COONHCOCH.sub.2 CH(i-Bu)CO-L-Trp-NHMe;
.PHI.COONHCOCH.sub.2 CH(i-Bu)CO-L-Trp-NHMe;
CH.sub.3 COONMeCOCH.sub.2 CH(i-Bu)CO-L-Trp-NHMe; and
.PHI.OCOONEtCOCH.sub.2 CH(i-Bu)CO-L-Trp-NHMe.
The reverse hydroxamates and hydroxyureas of formulas 3 and 4 are
more stable biologically than the corresponding hydroxamates per
se. This has been confirmed in Carter, G.W., et al., J Pharmacol
Exo Ther (1991) 256:929-937; Jackson, W.P., et al., J Med Chem
(1988) 31:499-500; Young, P.R., et al., FASEB J (1991) 5:A1273;
Hahn, R.A., et al., J Pharmacol Ex Ther (1991) 256:94-102;
Tramposch, K.M., et al., Agents Actions (1990) 30:443-450;
Argentieri, D.C., et al.; Kimball, E., et al., 5th Int Conf
Inflammation Research Assoc., Whitehaven, Pa., Sep. 23-27, 1990,
Abstract 100; and Huang, F., et al., J Med Chem (1989)
32:1836-1842. Thus, while somewhat more complicated to synthesize,
these analogs offer physiological characteristics which are
advantageous in the applications of these compounds to therapy.
The reverse hydroxamates and hydroxyureas of the invention are
obtainable using the standard techniques of synthetic organic
chemistry (see Challis, B.C., et al., "Amides and Related
Compounds" in "Comprehensive Organic Chemistry," Barton, D., et
al., eds. (1979) 2:1036-1045), pergamon Press, Oxford, as further
described below.
With respect to starting materials, the components forming the
--NR.sup.3 --CHR.sup.4 COX moiety are readily available in the case
of tryptophan and its analogs as esters or amides. As set forth
above, many analogous fused bicyclo aromatic amino acids are
described by Greenstein and Winitz (supra). Amino acids
corresponding to those wherein R.sup.4 is 1-(2-methyl
naphthyl)methylene; 1-quinolyl-methylene; 1-naphthyl methylene;
1-isoquinolyl methylene; and 3-isoquinolyl methylene can be
prepared from the bicyclo aromatic methylene halides using the
acetamido malonic ester synthesis of amino acids, as is well
understood in the art. The methylene halides themselves can be
prepared from their corresponding carboxylic acids by reduction
with lithium aluminum hydride and bromination of the resulting
alcohol with thionyl bromide.
Depending on the functional group symbolized by Y, the stage of
synthesis at which this moiety is brought into the compound of the
invention varies.
For those embodiments wherein Y is R.sup.7 ONR.sup.6 CONR.sup.6 --
and wherein n=0, 1 or 2, the compounds are prepared by acylating an
.alpha., .beta. or .gamma. amino acid, respectively with methyl or
ethyl chloroformate, condensing the resulting amino acid with a
protected form of the moiety --NR.sup.3 CHR.sup.4 COX and reacting
the resulting carboethoxy "dipeptide" with hydroxylamine or a
substituted hydroxylamine as described by Fieser, L.F., et al.,
"Reagents for Organic Synthesis" (1967) 1:479 (John Wiley &
Sons, New York). This sequence of reactions is shown in Reaction
Scheme 1A. ##STR8##
Alternatively, the .alpha., .beta. or .gamma. amino acid is
temporarily protected using, for example, carbobenzoxy or tertiary
butyloxycarbonyl and coupling it to the carboxy-terminal-protected
amino acid moiety containing R.sup.4. The protecting group is then
removed by hydrogenolysis or acidolysis as appropriate, and the
deprotected .alpha., .beta. or .gamma. amino group is reacted with
an activated carbonic acid such as carbonyldiimidazole. The
resultant is then reacted with hydroxylamine or substituted
hydroxylamine to obtain the desired product. This sequence of
reactions is summarized in Reaction Scheme 2. (In the formula
Im-Co-Im, Im represents an imidazole residue.) ##STR9##
The appropriate .alpha., .beta. or .gamma. amino acids are prepared
by general methods as set forth by Jones, J.H., et al., in "Amino
Acids," p. 834 (Barton, D., et al., eds.) ("Comprehensive Organic
Chemistry" (1979) Vol. 2, Pergamon Press). Such methods include,
for example, homologation by Arndt-Eistert synthesis of the
corresponding N-protected .alpha.-amino acid and more generally the
addition of nitrogen nucleophiles such as phthalimide to
.alpha.,.beta.-unsaturated esters, acids or nitriles.
In a second class of hydroxyureas, Y has the formula R.sup.6.sub.2
NCONOR.sup.7 -- and n is 0, 1 or 2. These compounds are prepared
from the corresponding .alpha., .beta. or .gamma. hydroxyamino
acids of the formula R.sup.7 ONH(CHR.sup.1).sub.n CHR.sup.2 COOH.
When both R.sup.6 are H, this intermediate is converted to the
desired hydroxyurea by reaction with silicon tetraisocyanate, as
described by Fieser and Fieser, "Reagents for Organic Synthesis"
(1968) 1:479 (John Wiley & Sons, New York). The reaction is
conducted with the hydroxyl group protected or substituted by
R.sup.7. The resulting hydroxyurea is then coupled to the component
of the formula HNR.sup.3 CHR.sup.4 COX to obtain the desired
product. Alternatively, the amide is first formed and the
N-hydroxyl dipeptide is treated with the reagent.
Alternatively, when Y is R.sup.6.sub.2 HNCO--NOR.sup.7, wherein
R.sup.6 is alkyl, the above O-protected .alpha., .beta. or .gamma.
N-hydroxyamino acid is reacted with the relevant alkylisocyanate
R.sup.6 NCO to produce the desired product.
When Y is of the formula R.sup.6.sub.2 NCO--NOR.sup.7 -- wherein
both R.sup.6 are alkyl, the .alpha., .beta. or .gamma.
N-hydroxyamino acid is reacted with an activated form of carbonic
acid, for example, carbonyldiimidazole or
bis-p-nitrophenylcarbonate, and then with the diamine R.sup.6.sub.2
NH wherein both R.sup.6 are alkyl groups. This is followed by
deprotection, if desired.
Conditions for the foregoing can be found in the descriptions of
analogous preparations for tripeptides as described by Nishino, N.,
et al., Biochemistry (1979) 18:4340-4346.
The .beta.-N-hydroxyamino acids used as intermediates in the
foregoing synthesis can be prepared by a malonic ester synthesis in
which diethyl malonate is alkylated twice, one with R.sup.2 --Br
and then with benzylchloromethyl ether, for example, for the case
wherein R.sup.1 is H. The product is saponified, decarboxylated,
hydrogenated, and oxidized to give the .beta.-aldehyde in a manner
similar to the synthesis of a homologous aldehyde described by
Kortylewicz, Z.P., et al., Biochemistry (1984) 23:2083-2087. The
desired .beta.-hydroxyamino acid is then obtained by addition of
protected (or alkylated, if R.sup.7 is alkyl or acylated if R.sup.7
is acyl) hydroxylamine. The corresponding compound wherein R.sup.1
is alkyl can be prepared in an analogous manner wherein the second
alkylation utilizes benzyl-O-CHR.sup.2 Cl. The homologous ketone
was described by Galardy, R.E., et al., Biochemistry (1985)
24:7607-7612.
Finally, those compounds wherein Y is of the formula R.sup.6
CONOR.sup.7 --, i.e., the reverse hydroxymates, can be prepared by
acylation of the corresponding .alpha., .beta. or .gamma. N-hydroxy
dipeptide. Alternatively, the N-hydroxyamino acid can be acylated,
followed by condensation to form the amide bond in the compounds of
the invention. The acylation method is described by, for example,
Nishino, N., et al., Biochemistry (1979) 18:4340-4346, cited
above.
Alternatively, for those compounds wherein n=1 and R.sup.1 is H,
the compounds can be prepared by condensing the ylide
1,1-dimethoxy-2-(triphenylphosphoranylidene) ethane prepared from
triphenylphosphine and 1,1-dimethoxy-2-bromoethane with
4-methyl-2-oxopentanoic acid. The product is then hydrogenated to
obtain 4,4-dimethoxy-2-isobutylbutanoic acid which is coupled to
the moiety R.sup.3 NHCHR.sup.4 COX to obtain
4,4-dimethoxy-2-isobutylbutanoyl--NR.sup.3 CHR.sup.4 COX. Treatment
with aqueous acid yields the aldehyde
2-isobutyl-4-oxobutanoyl--NR.sup.3 CHR.sup.4 COX. The oxime is
prepared by reaction with hydroxylamine and reduced to the
corresponding N-substituted hydroxylamine. Acylation of both the
hydroxaminol oxygen and nitrogen followed by hydrolysis of the
O-acyl group provides the N-acyl reverse hydroxymates. (Summers,
J.B., et al., J Med Chem (1988) 31:1960-1964.)
For compounds wherein --CONR.sup.3 -- is in modified isosteric
form, these forms can be prepared by methods known in the art, as
set forth above.
Preferred compounds of formulas (3) and (4) include:
EtONHCONMe-CH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
EtCONOH-CH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
n-PrCONOEt-CH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
EtNHCONOMe-CH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
MeNHCONOH-CH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
EtONHCONMe-CH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
EtCONOH-CH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
n-PrCONOEt-CH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
EtNHCONOMe-CH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
MeNHCONOH-CH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
HONHCONHCH.sub.2 CH(iBu)-CO-L-TrpNHMe;
HONHCONHCH.sub.2 CH.sub.2 CH(iBu)-CO-L-TrpNHMe;
HONHCONHCH(iBu)CO-L-TrpNHMe;
H.sub.2 NCON(OH)CH(iBu)CO-L-TrpNHMe;
N(OH)CH.sub.2 CH(iBu)CO-L-TrpNHMe;
H.sub.2 NCON(OH)CH.sub.2 CH.sub.2 CH(iBu)CO-L-TrpNHMe;
CH.sub.3 CON(OH)CH(iBu)CO-L-TrpNHMe;
CH.sub.3 CON(OH)CH.sub.2 CH(iBu)CO-L-TrpNHMe; and
CH.sub.3 CON(OH)CH.sub.2 CH.sub.2 CH(iBu)CO-L-TrpNHMe.
Administration and Use
Compounds which are synthetic inhibitors of mammalian
metalloproteases are useful to inhibit angiogenesis. These
compounds can therefore be formulated into pharmaceutical
compositions for use in inhibiting angiogenesis in conditions
characterized by an unwanted level of such blood vessel growth.
Standard pharmaceutical formulation techniques are used, such as
those disclosed in Remington's Pharmaceutical Sciences, Mack
Publishing Company, Easton, Pa., latest edition.
For indications to be treated systemically, it is preferred that
the compounds be injected. These conditions include tumor growth
and metastasis. The compounds can be formulated for injection using
excipients conventional for such purpose such as physiological
saline, Hank's solution, Ringer's solution, and the like. Injection
can be intravenous, intramuscular, intraperitoneal or subcutaneous.
Dosage levels are of the order of 0.1 .mu.g/kg of subject to 1
mg/kg of subject, depending, of course, on the nature of the
condition, the nature of the subject, the particular embodiment of
the invention compounds chosen, and the nature of the formulation
and route of administration.
In addition to administration by injection, the compounds of the
invention can also be formulated into compositions for transdermal
or transmucosal delivery by including agents which effect
penetration of these tissues, such as bile salts, fusidic acid
derivatives, cholic acid, and the like. The compounds can also be
used in liposome-based delivery systems and in formulations for
topical and oral administration depending on the nature of the
condition to be treated. Oral administration is especially
advantageous for those compounds wherein the moiety --CONR.sup.3 --
is in a modified isosteric form. These compounds resist the
hydrolytic action of the digestive tract. Oral formulations include
syrups, tablets, capsules, and the like, or the compound may be
administered in food or juice.
The inhibitors of the invention can be targeted to specific
locations where vascularization occurring is accumulated by using
targeting ligands. For example, to focus the compounds to a tumor,
the inhibitor is conjugated to an antibody or fragment thereof
which is immunoreactive with a tumor marker as is generally
understood in the preparation of immunotoxins in general. The
targeting ligand can also be a ligand suitable for a receptor which
is present on the tumor. Any targeting ligand which specifically
reacts with a marker for the intended target tissue can be used.
Methods for coupling the compounds to the targeting ligand are well
known and are similar to those described below for coupling to
carrier. The conjugates are formulated and administered as
described above.
For localized conditions, topical administration is preferred. For
example, to treat diabetes-induced retinopathy or other neovascular
glaucomas, direct application to the affected eye may employ a
formulation as eyedrops or aerosol. For this treatment, the
compounds of the invention can also be formulated as gels or
ointments, or can be incorporated into collagen or a hydrophilic
polymer shield. The materials can also be inserted as a contact
lens or reservoir or as a subconjunctival formulation.
In all of the foregoing, of course, the compounds of the invention
can be administered alone or as mixtures, and the compositions may
further include additional drugs or excipients as appropriate for
the indication.
Conditions that benefit from angiogenesis inhibition thus include,
generally, cancer, including angiosarcoma, Kaposi's sarcoma,
glioblastoma multiforme, hemangio blastoma, including von
Hippel-Lindan disease and hemangio pericytoma; eye conditions, such
as diabetic retinopathy and neovascular glaucoma; immune system
conditions, such as rheumatoid arthritis, angiolymphoid hyperplasia
with eosinophilia; and skin conditions, such as cavernous
hemangioma (including Kasabach-Merritt syndrome) and psoriasis.
The following examples are intended to illustrate but not to limit
the invention. These examples describe the preparation of certain
compounds of the invention and their activity in inhibiting
mammalian metalloproteases.
In the examples below, TLC solvent systems are as follows: (A)
ethyl acetate/methanol (95:5); (B) ethyl acetate/methanol (25:5);
(C) ethyl acetate; (D) ethyl acetate/methanol (30:5); (E) ethyl
acetate/hexane (1:1); (F) chloroform/methanol/acetic acid (30:6:2);
(G) chloroform/methanol/acetic acid (85:10:1).
EXAMPLE 1
Preparation of
N-[D,L-2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-tryptophan
methylamide
A suspension of 5 g (0.033 mol) of the sodium salt of
4-methyl-2-oxopentanoic acid and 5.65 g (0.033 mol) of benzyl
bromide in 10 ml of anhydrous dimethylformamide was stirred for 4
days at room temperature. After evaporation of the solvent under
reduced pressure the residue was diluted to 100 ml with hexane and
washed with water (3.times.20 ml) and saturated sodium chloride and
dried over anhydrous magnesium sulfate. Evaporation of solvent gave
6.4 g (88% yield) of the benzyl ester of 4-methyl-2-oxopentanoic
acid (1) as a colorless oil.
A mixture of 6.4 g (0.029 mol) of (1) and 9.7 g (0.029 mol) of
methyl(triphenylphosphoranylidene)acetate in 100 mL of dry
methylene chloride was stirred for 12 hr at room temperature and
evaporated to dryness. The residue was extracted with hexane
(3.times.50 mL). The hexane solution was washed with 10% sodium
bicarbonate (2.times.30 mL), water and saturated sodium chloride
and dried over anhydrous magnesium sulfate. Evaporation of the
solvent gave 8.01 g (100% yield) of benzyl
2-isobutyl-3-(methoxycarbonyl)-propionate (2) as a mixture of E and
Z isomers.
A mixture of 8.01 g (0.029 mol) of (2) and 1 g of 10% palladium on
carbon in 50 mL of methanol was hydrogenated at room temperature
under 4 atmospheres of hydrogen gas for 8 hr. After removal of the
catalyst by filtration the filtrate was evaporated to dryness under
reduced pressure to give 4.7 g (86% yield) of
2-iso-butyl-3-(methoxycarbonyl)-propionic acid (3) as a colorless
oil.
To a mixture of 0.85 g (4.5 mmol) of (3) and 0.57 g (4.5 mmol) of
oxalyl chloride in 10 mL of dry methylene chloride 0.1 mL of
anhydrous dimethylformamide was added. After stirring for 1 hr at
room temperature the solvent was evaporated under reduced pressure
and the residue was diluted to 5 mL with anhydrous
dimethylformamide and 1.06 g (4.1 mmol) of the hydrochloride salt
of L-tryptophan methylamide (Kortylewicz and Galardy, J Med Chem
(1990) 33:263-273) was added followed by addition of 1.3 mL (9.3
mmol) of triethylamine at -10.degree. C. This was stirred for 7 hr
at room temperature and evaporated to dryness at room temperature
under reduced pressure. The residue was diluted to 150 mL with
ethyl acetate and washed with water (2.times.15 mL), 10% potassium
bisulfate (5.times.20 mL), 10% sodium bicarbonate (2.times.20 mL),
saturated sodium chloride and dried over anhydrous magnesium
sulfate and then evaporated to give 1.6 g (83% yield) of
N-[D,L-2-isobutyl-3-(methoxycarbonyl)-propanoyl]-L-tryptophan
methylamide 4 as a mixture of diastereomers, 4A and 4B.
Isomers 4A and 4B were separated by flash chromatography (silica
gel, ethyl acetate).
Isomer 4A: mp=134.degree.-137.degree. C. Rf(C)=0.37.
Isomer 4B: mp=156.degree.-158.degree. C. Rf(C)=0.2.
Alternatively, the mixture of 4A and 4B was converted directly to
its hydroxamate as described below. In this case, 5A was
crystallized from the mixture of 5A and 5B.
A warm mixture of 0.22 g (3.96 mmol) of potassium hydroxide in 1 mL
of methanol was added to a warm mixture of 0.184 g (2.65 mmol) of
the hydrochloride salt of hydroxylamine. After cooling in ice under
an argon atmosphere the potassium chloride was filtered off and 0.5
g (1.32 mmol) of (4A) was added to the filtrate. The resulting
mixture was stirred for 7 hr at room temperature and evaporated to
dryness under reduced pressure. The residue was suspended in 100 mL
of ethyl acetate and washed with 10 mL of 10% potassium bisulfate,
saturated sodium chloride and dried over anhydrous magnesium
sulfate and evaporated to dryness under reduced pressure. The
residue was crystallized from ethyl acetate to give 0.28 g (56%
yield) of pure 5A.
Isomer 4B was converted to its corresponding hydroxamic acid 5B
(72% yield) as described for 4A.
Isomer 5A: mp=176.degree.-182.degree. C.. Rf(D)=0.45.
Isomer 5B: mp=157.degree.-162.degree. C. Rf(D)=0.39.
For the case wherein the 4A/4B mixture is used, the 5A can be
crystallized directly from the residue as described above.
In a similar manner to that set forth above, but substituting for
4-methyl-2-oxopentanoic acid, 2-oxopentanoic acid,
3-methyl-2-oxobutyric acid, 2-oxohexanoic acid,
5-methyl-2-oxohexanoic acid, or 2-decanoic acid, the corresponding
compounds of formula 1 are prepared wherein R.sup.1 is H and
R.sup.2 is an n-propyl, i-propyl, n-butyl, 2-methylbutyl, and
n-octyl, respectively. In addition, following the procedures set
forth hereinabove in Example 1, but omitting the step of
hydrogenating the intermediate obtained by the Wittig reaction, the
corresponding compounds of formula 2 wherein R.sup.1 is H and
R.sup.2 is as set forth above are obtained.
To synthesize the compounds containing acylated forms of the
indolyl residue, the intermediate ester of formula 3 or 4 is
deesterified and acylated prior to conversion to the hydroxamate.
For illustration, 4A is deesterified with sodium hydroxide in
ethanol and then acidified to give
N-(L-2-isobutyl-3-carboxypropanoyl)-L-tryptophan methylamide, which
is treated with the anhydride of an alkyl (1-4C) carboxylic acid to
obtain
N-(L-2-isobutyl-3-carboxypropanoyl)-L-((N-acyl)indolyl)tryptophan
methylamide. This intermediate is then treated with oxalyl chloride
followed by hydroxylamine at low temperature to give the
corresponding hydroxamate.
EXAMPLE 2
Preparation of
N-[2-isobutyl-3-(N'-hydroxycarbonylamido)-orgoanoyl]-D-tryptophan
methylamide (7B)
The mixture of the two diastereoisomers of
N-[2-isobutyl-3-(methoxycarbonyl)-propanoyl]-D-tryptophan methyl
amide 6A,B was prepared as described for 4A,B in Example 1. The
mixture was crystallized from ethyl acetate to give, after two
recrystallizations, 0.26 g (49%) of the pure diastereomer 6B: mp
155.degree.-157.degree. C., R.sub.f (C)=0.32. 6B was converted into
by the method described in Example 1 in 50% yield (119 mg): mp
157.degree.-159.degree. C., R.sub.f (D)=0.39.
EXAMPLE 3
Preparation of N-[2-isobutyl-3-(N'-
hydroxycarbonylamido)-propanoyl]-N-methyl-L-tryptophan methylamide
(9A)
The reaction of N-methyl-L-tryptophanmethylamide, prepared as
described in Example 1 for L-tryptophan methylamide, with 3
performed as described for 4 gave crude
N-[D,L-2-isobutyl-3-(methoxycarbonyl)-propanoyl]-N-methyl-L-tryptophan
methylamide 8A,B which was crystallized from ethyl acetate to give
76 mg (19% yield) of 8A: mp 171.degree.-174.degree. C., R.sub.f
(C)=0.40.
8A was converted into 9A by the method described in Example 1 in
45% yield (34 mg): mp 180.degree.-183.degree. C., R.sub.f
(D)=0.54.
EXAMPLE 4
Preparation of N-[2-isobutyl-3-(N-hydroxycarbonyl
amido)-propanoyl]-L-3-(2-naphthyl)-alanine methylamide (11A)
N-[D,L-isobutyl-3-(methoxycarbonyl)-propanoyl]-L-(3-(2-naphthyl)-alanine
10A was prepared as described in Example 1 from
L-3-(2-naphthyl)-alanine methylamide and 3. The crude product was
chromatographed on 60 g of silica gel in ethyl acetate:hexane 1:1
to yield 12 mg (5% yield) of 10A: mp 151.degree.-158.degree. C.,
R.sub.f (C)=0.69.
10A was converted into the hydroxamate 11A as in Example 1 in 30%
yield (3 mg): mp 179.degree.-181.degree. C., R.sub.f (D)=0.17.
MS-FAB (m/z) 400 (M.sup.+ +H)
EXAMPLE 5
Preparation of N-[2-isobutyl-3-(N'-hydroxycarbonyl
amido)-propanoyl]-L-tryptophan 2-hydroxyethylamide (13A)
The hydrochloride salt of L-tryptophan 2-hydroxyethylamide was
prepared and coupled with 3 as described for the hydrochloride salt
of L-tryptophan methylamide in Example 1 except that 3 was
activated with 1,1'-carbonyldiimidazole for 20 minutes in methylene
chloride at room temperature. The crude product was a mixture of
0.7 g (67% yield) of the diastereoisomers 12A,B: R.sub.f (C) 12A
0.38, R.sub.f (C) 12B 0.19.
12A crystallized from ethyl acetate in 35% yield (0.18 g): mp
161.degree.-163.degree. C., R.sub.f (C)=0.38.
12A was converted into
N-[2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-L-tryptophan
2-hydroxyethylamide 13A as in Example 1 in 35% yield (62 mg):
R.sub.f (D)=0.17, mp 162.degree.-163.degree. C. MS-FAB (m/z) 419
(M.sup.+ +H).
EXAMPLE 6
Preparation of N-[2-isobutyl-3-(N'-hydroxycarbonyl
amido)-orgoanoyl]-L-tryptophan amylamide (15A)
The hydrochloride salt of L-tryptophan amylamide was prepared as
described in Example 1 for L-tryptophan methylamide and was reacted
with 3 that had been activated with 1,1'-carbonyldiimidazole for 20
minutes in dichloromethane at room temperature. The mixture of the
two diastereomers of
N-[D,L-2-isobutyl-3-(methoxycarbonyl)-propanoyl]-L-tryptophan
amylamide 14A,B (90% yield) was converted to its corresponding
hydroxamic acids as described for 4A. Slow evaporation of the
ethylacetate solution gave 0.343 g (71%) of 15A,B: mp
160.degree.-163.degree. C. MS-FAB (m/z) 445 (M.sup.+ +H).
EXAMPLE 7
Preparation of N-2-isobutyl-3-(N'-hydroxycarbonyl
amido)-orgoanoyl]-L-tryptophan piperidinamide (17A,B)
L-tryptophan piperidinamide was reacted with 3 as performed in
Example 1 for L-tryptophan methylamide to give 1.4 g (89% yield) of
N-[D,L-2-isobutyl-3-(methoxycarbonyl)-propanoyl]-L-tryptophan
piperidinamide 16A,B as a foam; R.sub.f (C) (16A) 0.74, (16B)
0.67.
16A,B was converted into crude 17A,B identically to 4A in Example 1
in 88% yield (570 mg): R.sub.f (D) (17A) 0.41, (17B) 0.30. Crude
17A,B was chromatographed on 180 g of silica gel in 12% isopropanol
in ethyl acetate to give 140 mg (25% yield) of 17A,B after
crystallization from ethyl acetate: mp 169.degree.-170.degree. C.
MS-FAB (m/z) 443 (M.sup.+ +H).
EXAMPLE 8
Preparation of N-[2-isobutyl-3-(N'-hydroxycarbonyl
amido)-propanoyl]-L-tryptophan dodecylamide (19A)
The reaction of L-tryptophan dodecylamide was prepared in a manner
analogous to that described for L-tryptophan methylamide in Example
1. This ester was reacted with 3 as described in Example 1 to give
crude N-[D,L-isobutyl-3-(methoxycarbonyl)-propanol]-L-tryptophan
dodecylamide 18A,B in 93% yield as a mixture of isomers 19A and
19B. This mixture was chromatographed on 150 g of silica gel in
ethyl acetate:hexane, 1:2, to yield 0.62 g of the mixture of the
two isomers: R.sub.f (E) 19A 0.37, R.sub.f (E) 19B 0.29.
Crystallization by slow evaporation from ethyl acetate gave 0.38 g
of 18A contaminated by approximately 10% of 18B by TLC and NMR
analysis: mp 133.degree.-135.degree. C. 18A was converted to its
corresponding hydroxamic acid as described in Example 1, except
that the potassium salt of 19A crystallized from the alkaline
reaction mixture in 81% yield (222 mg). The potassium salt of 19A
(54 mg) was dissolved in 2 mL of boiling methanol, a few drops of
water were added, and the solution was acidified to pH 6 with 0.1 N
hydrochloric acid and diluted with water to give 50 mg (100% yield)
of 19A: mp 155.degree.-159.degree. C., R.sub.f (D)=0.49. MS-FAB
(m/z) 543 (M.sup.+ +H).
EXAMPLE 9
Preparation of N-[2-isobutyl-3-(N'-hydroxycarbonylamido)
propanoyl]-L-tryptophan (S)-methylbenzylamide (21A)
The reaction of L-tryptophan (S)-methylbenzylamide with 3 was
performed as described in Example 1 to give, after crystallization
from ethyl acetate, 330 mg (51% yield) of
N-[2-isobutyl-3-(methoxycarbonyl)-propanoyl]-L-tryptophan
(S)-methylbenzylamide 20A: mp 160.degree.-162.degree. C., R.sub.f
(C)=0.77.
20A was converted into hydroxamate 21A by the identical method used
in Example 1 in 38% yield (76 mg): mp 165.degree.-166.degree. C.,
R.sub.f (D)=0.73. MS-FAB (m/z) 479 (M.sup.+ +H).
EXAMPLE 10
Preparation of
N-[L-2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-L-tryptophan
(6-phenylmethoxycarbonylamino-hexyl-1)amide (27A)
To prepare 1-amino-6-phenylmethoxycarbonylaminohexane (23), an
equimolar mixture (0.01 mol) of 1,6-diaminohexane and benzaldehyde
in 25 mL of methylene chloride was stirred for 5 hr in the presence
of 1.5 g of anhydrous magnesium sulfate at room temperature. After
removing the drying agent by filtration the filtrate was evaporated
to dryness under reduced pressure to give 2 g (100% yield) of crude
1-amino-6-phenylamino-hexane 22 as a colorless oil; NMR(CDCl.sub.3)
1.1-1.9(m, 10H, hexane CH.sub.2 -2,-3,-4,-5, NH.sub.2); 2.6(m, 2H,
CH.sub.2 -1); 3.51(m, 2H, hexane CH.sub.2 -6); 7.1-7.8 (m, 5H,
aromatic); 8.16(s, 1H, imine CH). To a mixture of 2 g (0.01 mol) of
22 and 1.4 mL (0.01 mol) of triethylamine in 20 mL of methylene
chloride. Then 1.78 g (0.01 mol) of benzylchloroformate was added
dropwise at -5.degree. C. The resulting mixture was stirred for 0.5
hr at 0.degree. C. and for 2 hr at room temperature then diluted to
50 mL with methylene chloride and washed with water (20 ml), 2%
sodium bicarbonate (20 ml), water and saturated sodium chloride and
dried over anhydrous magnesium sulfate. After evaporation of
solvent under reduced pressure the residue was dissolved in 5 mL of
ethanol and 10 mL of 2N hydrochloric acid was added. The resulting
mixture was stirred for 6 hr at room temperature then evaporated to
dryness under reduced pressure. The residue was diluted to 50 mL
with water and washed with ethyl ether (2.times.15 ml). The water
phase was evaporated under reduced pressure and the product 23 was
purified by crystallization from a small portion of water with a
yield of 42%; mp 175.degree.-178.degree. C.
To prepare the dipeptide analog
(N-(L-2-isobutyl-3-methoxycarbonyl)-propanoyl-L-tryptophan (25A)),
for derivatization to 23: To a mixture of 1.754 g (9.32 mmol) of
2-isobutyl-3-methoxycarbonylpropionic acid 3 in 4 mL of 50%
anhydrous DMF in methylene chloride 1.66 g (10.2 mmol) of
N,N'-carbonyldiimidazole (CDI) was added at room temperature. After
15 minutes of stirring at room temperature, 3.08 g (9.31 mmol) of
the hydrochloride salt of L-tryptophan benzyl ester was added. The
resulting mixture was stirred overnight at room temperature, then
diluted to 60 mL with ethyl acetate and washed with 5% sodium
bicarbonate (2.times.15 ml), water (2.times.15 ml), saturated
sodium chloride solution and dried over magnesium sulfate.
Evaporation of the solvent under reduced pressure gave 4.32 g (100%
yield) of 24, the benzyl ester of 25 as a colorless foam, which was
used in the next step without further purification.
Hydrogen gas was bubbled through a mixture of 4.32 g (9.31 mmol) of
24 and 0.5 g of 10% palladium on carbon in 15 mL of methanol for 2
hr while methanol was added to keep the volume of the reaction
mixture constant. The catalyst was filtered off and washed with a
fresh portion of methanol (15 ml) and the filtrate was evaporated
to dryness under reduced pressure. Evaporation of the solvent under
reduced pressure and drying of the residue in vacuo gave 3.08 g
(88% yield) of acid 25A,B as a mixture of two diastereoisomers, in
the form of a colorless glassy solid. This was separated to give
isomers 25A and 25B by flash chromatography (silica gel; ethyl
acetate; R.sub.f (25A)=0.24, R.sub.f (25B)=0.1).
The compound 25A was converted to
N-[L-2-isobutyl-3-methoxycarbonylpropanoyl]-L-tryptophan
(6-phenylmethoxycarbonylamino-hexyl-1)amide (26A) as follows. A
mixture of 0.55 g (1.47 mmol) of 25A and 0.24 g (1.48 mmol) of CDI
in 1 mL of 2% dimethylformamide in methylene chloride was stirred
for 0.5 hr at room temperature and 0.42 g (1.47 mmol) of 23 was
added. After stirring overnight at room temperature, the mixture
was diluted to 50 mL with chloroform and washed with 2% potassium
bisulfate (2.times.10 ml), water (10 ml), 5% sodium bicarbonate
(2.times.10 ml), water (2.times.10 ml) and saturated sodium
chloride and dried over anhydrous magnesium sulfate. Evaporation of
the solvent under reduced pressure gave 0.8 g of the crude 26A
which was purified by flash chromatography (silica gel; ethyl
acetate/hexane 25:5): Yield 56%; R.sub.f (E)=0.57.
When the product 26A is substituted for 4A in Example 1, the
identical process afforded the title compound 27A, melting at
102.degree.-108.degree. C., in 46% yield; R.sub.f (D)=0.63.
EXAMPLE 11
Preparation of
N-[L-2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-L-tryptophan
cyclohexylamide (28A)
When cyclohexylamine is substituted for 23 in Example 10, the
identical process afforded the title compound 28A melting at
199.degree.-203.degree. C., in 49% yield; R.sub.f (D)=0.51.
EXAMPLE 12
Preparation of
N-[cis-2-(N'-hydroxycarbonylamido)-cyclohexylcarbonyl]-L-tryptophan
methylamide (29A,B)
A mixture of 2 g (0.013 mol) of cis-1,2-cyclohexane-dicarboxylic
anhydride in 15 mL of methanol was refluxed for 5 hr, then
evaporated to dryness under reduced pressure to give 2.41 g (100%
yield) of cis-2-methoxycarbonyl-cyclohexanecarboxylic acid. When
this was substituted for 3 in Example 1, the identical process
afforded the title compound, melting at 140.degree.-144.degree. C.,
in 36% yield; R.sub.f (D)=0.53, 0.47.
EXAMPLE 13
Preparation of
N-trans-2-(N'-hydroxycarbonylamido)-cyclohexylcarbonyl]-L-tryptophan
methylamide (30A,B)
When (.+-.)trans-1,2-cyclohexanedicarboxylic anhydride was
substituted for cis-1,2-cyclohexanedicarboxylic anhydride in
Example 12, the identical process afforded the title compound
30A,B, melting at 167.degree.-174.degree. C., in 37% yield; R.sub.f
(D)=0.57.
EXAMPLE 14
Preparation of
N-[2-isobutyl-3-(N'-hydroxycarbonylamido)-propanoyl]-L-tryptophan
(31A)
31A was prepared from 25A in Example 10 in a similar manner to the
preparation of 5A in Example 1 in 75% yield (128 mg) and isolated
as a foam from ethyl acetate: R.sub.f (F)=0.55, MS-FAB (m/z)
(M.sup.+ +H). A small sample of 31A recrystallized from ethyl
acetate had a melting point of 116.degree.-120.degree. C.
EXAMPLE 15
Preparation of N-(D,L-2-isobutyl-3-carboxypropanoyl)-L-tryptophan
(6-aminohexyl-1)amide (32A)
A mixture of 0.5 g (8.24 mmol) of 26A in 0.4 mL of 2N potassium
hydroxide in methanol was stirred overnight at room temperature,
then evaporated to dryness under reduced pressure. The residue was
diluted to 15 mL with water and acidified to pH =2 with 1N
hydrochloric acid. The crude free acid of 26A was taken up with
ethyl acetate (3.times.15 ml) and the organic phase was dried over
anhydrous magnesium sulfate and evaporated to dryness to give 0.45
g (92% yield) of 26A as a colorless foam.
Hydrogen gas was bubbled through a mixture of 0.395 g (6.6 mmol) of
the free acid of 26A in 15 mL of methanol for 2 hr, in the presence
of 0.12 g of 10% palladium on carbon at room temperature. The
catalyst was filtered off, washed with ethanol (2.times.20 ml) and
the filtrate was evaporated to dryness under reduced pressure to
give 0.3 g (92% yield) of the title compound 32A as a colorless
foam; R.sub.f (G) =0.08.
EXAMPLE 16
Preparation of
N-[N-(2-isobutyl-3-carboxypropanoyl)-L-tryptophanyl]glycine
34A,B
The reaction of L-tryptophanyl-glycine methyl ester with acid 3,
performed as described for 25A gave crude
N-[N-(D,L-2-isobutyl-3-methoxycarbonylpropanoyl)-L-tryptophanyl]-glycine
methyl ester 33 in 87% yield as a mixture of diastereomers 33A and
33B. Isomers 33A and 33B were separated by flash chromatography
(silica gel; ethyl acetate). Isomer 33A mp=154.degree.-155.degree.
C.; R.sub.f (C) =0.46.
Esters 33A,B were transformed to free acids 34A,B by saponification
with two equivalent of methanolic potassium hydroxide, as described
for 25A. Isomer 34A yield 92%; mp=96.degree.-102.degree. C.;
R.sub.f (G)=0.31.
Isomer 34B yield 93%; mp=99-105.degree. C.; R.sub.f (G)=0.25.
EXAMPLE 17
Preparation of N-(cis-2-carboxy-cyclohexylcarbonyl)-L-tryptophan
methylamide 35
To a mixture of 0.281 g (1.82 mmol) of
cis-1,2-cyclohexanedicarboxylic anhydride and 0.47 g of the
hydrochloride salt of L-Trp-NHMe in 0.5 mL of dimethylformamide
0.51 mL of triethylamine was added at room temperature. After 2 hr
of stirring the resulting mixture was diluted to 10 mL with water
and 25 mL of ethyl acetate was added. The resulting mixture was
acidified to pH=2 with 10% potassium bisulfate and the organic
phase was washed with water (2.times.15 ml), saturated sodium
chloride and dried over anhydrous magnesium sulfate and evaporated
to dryness. The title compound 35 was purified by crystallization
from an ethyl acetate-hexane mixture. Yield 48%;
mp=105.degree.-112.degree. C.; R.sub.f (G)=0.65, 0.61.
EXAMPLE 18
Preparation of N-(trans-2-carboxy-cyclohexylcarbonyl)-L-tryptophan
methylamide 36
When (.+-.) trans-1,2-cyclohexanedicarboxylic anhydride is
substituted for cis-1,2-cyclohexanedicarboxylic anhydride in
Example 17, the identical process afforded the title compound 36 in
56% yield: mp=167.degree.-174.degree. C.; R.sub.f (G)=0.67,
0.61.
EXAMPLE 19
Preparation of
N-[2-isobutyl-3-(N'-acetoxycarbonylamido)propanoyl]-L-tryptophan
methylamide (37A)
To 97.5 mg (0.25 mmol) of 5A (Example 1) in 0.5 ml of
dimethylformamide was added 25.5 mg (0.25 mmol) of acetic anhydride
and 37 mg (0.25 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
at room temperature. After standing overnight, the DMF was
evaporated under high vacuum and the residue taken up in a mixture
of equal volumes of ethyl acetate and 2% potassium bisulfate. The
ethyl acetate layer was washed with 2% potassium bisulfate, water,
and brine, dried over magnesium sulfate, and evaporated to give a
solid. The solid was dissolved in a 1:1 mixture of hot ethyl
acetate:hexane, which upon standing at room temperature gave 71 mg
(66% yield) of solid product 37A: mp=184.degree.-186.degree. C.;
R.sub.f (G)=0.68.
EXAMPLE 20
Preparation of
N-isobutyl-3-(N'-benzoxycarbonylamido)propanoyl]-L-tryptophan
methylamide (38A)
To 30.5 mg (0.25 mmol) of benzoic acid in 1 ml of tetrahydrofuran
was added 40.5 mg (0.25 mmol) of carbonyldiimidazole. After 10
minutes, 97 mg (0.25 mmol) of compound 5A from Example 1 was added
in 1 ml of dimethylformamide. After 10 minutes, the reaction
mixture was evaporated to dryness under high vacuum, and dissolved
in a mixture of equal volumes of ethyl acetate and water. The ethyl
acetate layer was washed with 5% sodium bicarbonate, water, 2%
sodium bisulfate, water, and brine, and dried over magnesium
sulfate. Evaporation of the ethyl acetate layer to a small volume
gave 50 mg (41%) of the title compound, 38A:
mp=187.degree.-187.5.degree. C.; Fr(G)=0.54.
EXAMPLE 21
Applying the methods set forth above, the following invention
compounds are synthesized:
HONHCOCH.sub.2 CH(n-hexyl)-CO-L-Trp-NHMe;
HONHCOCH.sub.2 CH(n-pentyl)-CO-L-Trp-NHMe;
HONHCOCH.sub.2 CH(i-pentyl)-CO-L-Trp-NHMe;
HONHCOCH.sub.2 CH(ethyl)-CO-L-Trp-NHMe;
HONHCOCH.sub.2 CH(ethyl)-CO-L-Trp-NHCH.sub.2 CH.sub.3 ;
HONHCOCH.sub.2 CH(ethyl)-CO-L-Trp-NHCH.sub.2 CH.sub.2 OH;
HONHCOCH.sub.2 CH(ethyl)-CO-L-Trp-NHcyclohexyl;
MeONHCOCH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
EtONMeCOCH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
MeONHCOCH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
EtONMeCOCH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
EtONHCONMe-CH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
EtCONOH-CH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
n-PrCONOEt-CH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
EtNHCONOMe-CH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
MeNHCONOH-CH.sub.2 CH(iBu)-CO-L-Trp-NHEt;
EtONHCONMe-CH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
EtCONOH-CH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
n-PrCONOEt-CH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
EtNHCONOMe-CH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
MeNHCONOH-CH.sub.2 CH(iBu)-CO-L-Ala(2-naphthyl)-NHEt;
HONHCONHCH.sub.2 CH(iBu)-CO-L-TrpNHMe;
HONHCONHCH.sub.2 CH.sub.2 CH(iBu)-CO-L-TrpNHMe;
HONHCONHCH(iBu)CO-L-TrpNHMe;
H.sub.2 NCON(OH)CH(iBu)CO-L-TrpNHMe;
N(OH)CH.sub.2 CH(iBu)CO-L-TrpNHMe;
H.sub.2 NCON(OH)CH.sub.2 CH.sub.2 CH(iBu)CO-L-TrpNHMe;
CH.sub.3 CON(OH)CH(iBu)CO-L-TrpNHMe;
CH.sub.3 CON(OH)CH.sub.2 CH(iBu)CO-L-TrpNHMe; and
CH.sub.3 CON(OH)CH.sub.2 CH.sub.2 CH(iBu)CO-L-TrpNHMe.
Determination of the inhibitory activity of certain of the
compounds prepared is conducted as described above, and provides
the results shown in Table 1.
TABLE 1
__________________________________________________________________________
No. Compound K.sub.i (nM)
__________________________________________________________________________
1 5A NHOHCOCH.sub.2 CH(i-Bu)COLTrpNHMe 10 1 5B NHOHCOCH.sub.2
CH(i-Bu)COLTrpNHMe 150 2 7A NHOHCOCH.sub.2 CH(i-Bu)CODTrpNHMe
70,000 3 9A NHOHCOCH.sub.2 CH(i-Bu)COLNMeTrpNHMe 500 4 11A
NHOHCOCH.sub.2 CH(i-Bu)COLAla(2-naphthyl)NHMe 15 5 13A
NHOHCOCH.sub.2 CH(i-Bu)COLTrpNH(CH.sub.2).sub.2 OH 20 6 15A
NHOHCOCH.sub.2 CH(i-Bu)COLTrpNH(CH.sub.2).sub.4 CH.sub.3 30 7 17A,
B NHOHCOCH.sub.2 CH(i-Bu)COLTrppiperidine 200 8 19A NHOHCOCH.sub.2
CH(i-Bu)COLTrpNH(CH.sub.2).sub.11 CH.sub.3 300 9 21A NHOHCOCH.sub.2
CH(i-Bu)COLTrpNH(S)CHMePh 3 10 27A NHOHCOCH.sub.2
CH(i-Bu)COLTrpNH(CH.sub.2).sub.6 NHCBZ 13 11 28A NHOHCOCH.sub.2
CH(i-Bu)COLTrpNHcyclohexyl 50 12 29A, B ##STR10## >10,000 13
30A, B ##STR11## >10,000 14 31A NHOHCOCH.sub.2 CH(i-Bu)LTrpOH
200 15 32A HOOCCH.sub.2 CH(i-Bu)COLTrpNH(CH.sub.2)NH.sub.2
>10,000 16 34A HOCOCH.sub.2 CH(i-Bu)COLTrpGlyOH >10,000 34B
HOCOCH.sub.2 CH(i-Bu)COLTrpGlyOH >10,000 17 35 ##STR12##
>10,000 18 36 ##STR13## >10,000
__________________________________________________________________________
EXAMPLE 22
Inhibition of Angiogenesis
A crude extract (30 mg/mL protein) of Walker 256 carcinoma, a rat
malignant tumor, was incorporated into Hydron, a slow release
polymer, in 1.5 mm diameter pellets. Pellets were implanted in the
stroma of the corneas of anesthetized albino rats. A cannula was
chronically implanted in the inferior vena cava, through which 10
mg/mL of compound 5A in 55% DMSO in water was infused continuously
for six days at the rate of 0.8 mL/24 hr. Controls received only
the DMSO solution. After six days, the animals were re-anesthetized
and perfused intra-arterially with India ink in order to visualize
the corneal vessels. The eyes were then enucleated and fixed in 5%
formalin. Control eyes which received only the DMSO solution show
massive vessel growth toward the pellet from the limbus. The
animals receiving compound 5A show vessels much shorter and/or much
finer than in the controls, barely filling with ink.
* * * * *